Mouse F9 cells differentiate into primitive endoderm when treated with retinoic acid (RA) and into parietal endoderm when treated with RA and dibutyryl cAMP. Differentiation, marked by changes in morphology and cellular physiology, depends on numerous signals including those imparted by the Wnts. Wnt6 transfected into F9 cells causes P-catenin translocation to the nucleus and the formation of primitive endoderm. Constitutive activation of G al 3 also causes the P-catenin translocation, however, cells differentiate through primitive to parietal endoderm. We hypothesize that differentiation to primitive endoderm requires Wnt signaling, and a G protein-dependent pathway involving G al 3 is necessary for differentiation to parietal endoderm. To test this, we transfected a constitutively active G a l3 mutant into F9 cells and found that during differentiation it signals through pll5RhoG EF and RhoA. Inhibiting Rho Kinase blocks the G a l3, but not the Wnt6-mediated differentiation to primitive endoderm. G a l3, known to signal through RhoA to regulate the actin cytoskeleton, can activate members of the Band 4.1 superfamily of proteins that include ezrin, radixin and moesin (ERM). Knocking down the expression of moesin in F9 cells, by shRNA or a morpholino strategy, alters the distribution of actin and ERM proteins, which leads to apoptosis. The presence of the constitutively active G a l3 mutant in the moesin-depleted cells prevents apoptosis, but does not facilitate differentiation. Overexpression of moesin, however, induces differentiation of F9 cells into primitive endoderm. Together, results indicate that Wnt6 signaling is sufficient to promote primitive endoderm formation, but G a l3 activation is necessary to induce parietal endoderm.